CN106319069B

CN106319069B - Kit for accurately determining pathogenic cells and application thereof

Info

Publication number: CN106319069B
Application number: CN201610861587.4A
Authority: CN
Inventors: 杨国华; 吕娟; 陈倩; 徐景祥
Original assignee: Henan Universe Technology Co Ltd
Current assignee: Jiangsu Genuo Biotechnology Co ltd
Priority date: 2016-09-28
Filing date: 2016-09-28
Publication date: 2020-02-14
Anticipated expiration: 2036-09-28
Also published as: CN106319069A

Abstract

The invention relates to a kit for accurately determining pathogenic cells and application thereof. The invention provides a kit for detecting pathogenic cells, which comprises: and detecting targeting molecules and nonspecific signals of the pathogenic cells to monitor the oligonucleotide probes. The non-specific signal monitoring oligonucleotide probe can monitor a non-specific signal generated by the binding of the oligonucleotide portion of the targeting molecule to the cell. The method of the invention effectively reduces the interference caused by non-specific binding and improves the accuracy of detecting the pathogenic cells.

Description

Kit for accurately determining pathogenic cells and application thereof

Technical Field

The invention belongs to the field of diagnosis of pathogenic cells, and particularly relates to a kit for accurately determining pathogenic cells and application thereof.

Background

Quantitative detection of pathogenic cells is a direct evidence for accurate determination of the patient's condition and for giving immediate treatment. For example, the number of circulating tumor cells in the blood is an important factor in determining the prognosis of a patient. Similarly, the detection of plasmodium in blood is an important index for judging the malaria disease.

Conventional detection methods are based essentially on the detection and quantification of antigens on the surface of the pathogenic cells. In these methods, the antibody is first attached to a solid support, and the antibody and the cells to be detected form a complex due to the antibody-antigen interaction. In the same manner, the antibody may be first complexed with the cell to be tested by an antigen-antibody reaction and then attached to a solid support. Thereafter, the cells caught by the antibody are collected and labeled for quantification. Such as immunofluorescence techniques, to detect circulating tumor cells. Tumor cells are first enriched from blood with antibodies attached to magnetic beads or microfluidic chips. Then, the tumor cells are labeled by the antibody with the marker and the specificity of the tumor cells and are quantitatively detected. Generally, the label includes radioactive isotopes, dyes, fluoresceins, enzymes (for enzyme labeling reactions), and the like. Although detection methods based on antigen capture are widely used clinically, these methods have many disadvantages, including low detection sensitivity, low throughput, cumbersome operation, and large human error.

The inventor's earlier patent targeting molecule for detecting pathogenic cells and application thereof (application number 201110315281.6) provides a targeting molecule for detecting pathogenic cells, which has a structure of X-Y, wherein X represents a binding molecule for specifically targeting the pathogenic cells, Y represents an oligonucleotide probe, and the oligonucleotide probe is subjected to fluorescent quantitative PCR amplification to realize signal amplification, thereby improving the detection sensitivity of specific binding molecules on the surface of the pathogenic cells and achieving the purpose of quantifying the pathogenic cells to be detected. However, the inventors found in their studies that: (1) the product obtained by enriching the pathogenic cells contains other irrelevant cells in addition to the pathogenic cells. For example, after enriching Circulating Tumor Cells (CTCs) in a blood sample, they contain a large number of leukocytes in addition to the CTCs. In addition, the number of leukocytes is also significantly higher than normal for most benign patients due to the inflammatory response. Because the expression of DNA receptors may exist on the surfaces of these pathogenic cells and irrelevant cells, the DNA receptors can be combined with the oligonucleotide probe part of the targeting molecule to generate non-specific binding signals, thereby affecting the detection accuracy. (2) Due to sample quality problems, such as storage time of the blood sample exceeding 24 hours or improper storage temperature, cell clumping is easily caused, and non-specific binding to the oligonucleotide part of the targeting molecule is also increased.

Therefore, it is necessary to develop a new detection reagent or method to improve the specificity of detection.

Disclosure of Invention

The invention aims to provide a kit for accurately determining pathogenic cells and application thereof.

In a first aspect of the present invention, there is provided a kit for detecting a pathogenic cell, the kit comprising: detecting targeting molecules of pathogenic cells, wherein the structure is X-Y; wherein X represents a binding molecule that specifically targets the pathogenic cell; y represents an oligonucleotide probe; "-" represents a covalent linkage, coupling or coupling relationship between X and Y; and

the sequence length and sequence information of the non-specific signal-monitoring oligonucleotide probe Y ', Y' are similar to those of Y, but differ from those of Y in some bases in sequence information.

In a preferred embodiment, the difference in sequence length between Y 'and Y is less than or equal to 30%, and the GC% difference between the sequences of Y' and Y is less than or equal to 30%.

In another preferred embodiment, the sequence length of Y or Y' is 10-150bp (double-stranded) or 10-150nt (single-stranded).

In another preferred embodiment, the sequence length of Y' is 11-120bp (double-stranded) or 11-120nt (single-stranded); such as 12bp (double stranded) or 12nt (single stranded); 14bp (double stranded) or 14nt (single stranded); 16bp (double stranded) or 16nt (single stranded); 18bp (double stranded) or 18nt (single stranded); 20bp (double stranded) or 20nt (single stranded); 30bp (double-stranded) or 30nt (single-stranded); 40bp (double stranded) or 40nt (single stranded); 50bp (double stranded) or 50nt (single stranded); 60bp (double stranded) or 60nt (single stranded); 80bp (double stranded) or 80nt (single stranded); 100bp (double stranded) or 100nt (single stranded).

In another preferred embodiment, when the sequence length of Y or Y 'is 10-50bp (double-stranded) or 10-50nt (single-stranded), Y' differs from Y in sequence information by 2-6, preferably 3-5 bases; when the sequence length of Y or Y 'is 51-100bp (double-stranded) or 51-100nt (single-stranded), there is a difference of 3-30, preferably 5-20 bases between Y' and Y in the sequence information; when the sequence length of Y or Y 'is 101-150bp (double-stranded) or 101-150nt (single-stranded), there is a difference of 3-45, preferably 5-35 bases between Y' and Y in the sequence information.

In another preferred embodiment, in the kit, X is selected from: an antibody, ligand, chemical small molecule or polypeptide that specifically targets a pathogenic cell.

In another preferred embodiment, the pathogenic cell is a tumor cell, and X is folate or a folate-cysteine conjugate; more preferably, the tumor cell is a circulating tumor cell.

In another preferred embodiment, the oligonucleotide probe has a sequence in which a thio modification is present in a phosphate linkage.

In another preferred embodiment, the kit further comprises: an extension primer aiming at Y, wherein the sequence structure of the extension primer is as follows: A-B; wherein A is a random sequence, and the B sequence is complementary to the sequence at one end (e.g., 3' end) of the Y sequence; and an extension primer for Y', the extension primer having the following sequence structure: A-B'; wherein A is as defined above and the B ' sequence is complementary to the sequence at one of the termini (e.g., the 3 ' end) of the Y ' sequence.

In another preferred embodiment, the extension primer is provided when the oligonucleotide portion of the targeting molecule is short, e.g., <50 bp.

In another preferred embodiment, the kit further comprises: a detection reagent that specifically detects an extended sequence comprising the Y and Y' sequences.

In another preferred embodiment, the detection reagent for specifically detecting the extended sequence comprising the Y and Y' sequences is a fluorescent quantitative PCR detection reagent; preferably, the fluorescent quantitative PCR detection reagent comprises: a forward primer, a reverse primer and a specific fluorescent probe that specifically amplify the extended sequence comprising the Y and Y' sequences.

In another aspect of the invention, the use of said kit is provided for the non-diagnostic in vitro detection of pathogenic cells.

In another aspect of the invention, there is provided a method for non-diagnostically detecting a pathogenic cell in vitro, the method comprising:

(1) providing the kit, blending the targeting molecule and the nonspecific signal monitoring oligonucleotide probe Y' with a sample to be detected, and incubating;

(2) collecting cells after incubation, and separating (e.g. eluting) the targeting molecules and the non-specific signal monitoring oligonucleotide probes Y' from the cells respectively;

(3) performing PCR quantitative analysis by using the targeting molecules separated in the step (2) as templates to obtain a quantitative analysis result 1; performing PCR quantitative analysis by using the nonspecific signal monitoring oligonucleotide probe Y' separated in the step (2) as a template to obtain a quantitative analysis result 2;

(4) and (4) subtracting the quantitative value of the quantitative analysis result 2 from the quantitative value of the quantitative analysis result 1 obtained in the step (3) to obtain the existence condition and the existence quantity of the pathogenic cells in the sample to be detected.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIGS. 1, 3mL of healthy human blood samples, spiked with 20 and 0 KB cells, respectively, and labeled with oligonucleotide ligated or not ligated with folate, change in the detected values.

Detailed Description

The inventor of the invention has conducted intensive research and discloses a method for monitoring nonspecific signals in the process of targeted quantitative detection of pathogenic cells. The method of the invention comprises selecting an oligonucleotide fragment as a nonspecific signal monitoring oligonucleotide probe, the length and GC% of which are similar to those of the oligonucleotide probe part for detecting the targeting molecules of pathogenic cells (the difference is less than or equal to 30%), and the nonspecific signal generated by the combination of the oligonucleotide part of the targeting molecules and the cells can be monitored. The method of the invention effectively reduces the interference caused by non-specific binding and improves the accuracy of detecting the pathogenic cells.

Term(s) for

As used herein, the term "pathogenic cell" refers to a cell derived from a diseased tissue (fixed to or released from the diseased tissue and freely present in a body fluid), which has a specific molecule (e.g., a surface antigen or receptor) on its surface, which is specific for the diseased cell and can be bound by a specific binding molecule (e.g., an antibody, a ligand), which is a binding molecule that specifically targets the diseased cell. For example, folate can recognize and bind to a folate receptor (folate receptor) on the surface of cancer cells, and monoclonal antibodies against Her2 can recognize and bind to Her2 antigen on the surface of breast cancer cells; LHRH polypeptides can recognize and bind to the luteinizing hormone releasing hormone receptor (LHRH receptor) on the surface of prostate cancer cells, and the like.

As used herein, the "binding molecule" is capable of specifically targeting a particular molecule (e.g., a surface antigen or receptor) on a pathogenic cell, with high affinity, and with an equilibrium dissociation constant of typically less than 10^-6. Such as but not limited to antibodies, ligands, chemical small molecules or polypeptides.

As used herein, the term "non-specific signal monitoring oligonucleotide probe" refers to a specifically designed probe that has a sequence length and GC% similarity (difference ≦ 30%) to the sequence length and GC% of the oligonucleotide probe on the targeting molecule. Because the non-specific signal monitoring oligonucleotide probe has high similarity with the oligonucleotide probe on the targeting molecule, and the non-specific binding performance of the non-specific signal monitoring oligonucleotide probe and the oligonucleotide probe on the targeting molecule is similar on cells, the non-specific binding condition of the oligonucleotide probe on the targeting molecule can be known by examining the non-specific binding condition of the non-specific signal monitoring oligonucleotide probe on the cells, so that the interference of the non-specific binding on the detection result is eliminated.

As used herein, the "cancer" or "tumor" is not particularly limited, and preferably the "cancer" or "tumor" will disseminate cancer cells or tumor cells into the blood circulation from the onset. For example selected from (but not limited to): nasopharyngeal carcinoma, esophageal carcinoma, gastric carcinoma, liver carcinoma, breast carcinoma, colorectal carcinoma, prostate carcinoma, lung carcinoma, cervical carcinoma, leukemia, oral carcinoma, salivary gland tumor, malignant tumor of nasal cavity and paranasal sinuses, laryngeal carcinoma, ear tumor, eye tumor, thyroid tumor, mediastinal tumor, chest wall, pleural tumor, small intestine tumor, biliary tract tumor, pancreatic and peri-ampullar tumor, mesenteric and retroperitoneal tumor, kidney tumor, adrenal tumor, bladder tumor, prostate carcinoma, testicular tumor, penile cancer, endometrial carcinoma, ovarian malignant tumor, malignant trophoblastic tumor, vulval cancer and vaginal cancer, malignant lymphoma, multiple myeloma, soft tissue tumor, bone tumor, skin and accessory tumor, malignant melanoma, nervous system tumor, and infantile tumor. As a preferred embodiment of the present invention, said "cancer" or "tumor" is selected from: breast (adenocarcinoma) and lung cancer.

As used herein, the "equilibrium dissociation constant (Kd)" refers to the concentration of ligand, antibody, small chemical molecule or polypeptide required to occupy half of the cells for a particular receptor or surface molecule. The Kd value is in an inverse relation with the receptor affinity, and the smaller the Kd value is, the higher the affinity is shown; conversely, a larger Kd value indicates a lower affinity. Usually Kd is less than 10^-6M represents a receptor or surface molecule with which a ligand, antibody, chemical small molecule or polypeptide has high affinity.

As used herein, the term "specific" refers to an antibody, ligand, polypeptide, or small chemical molecule that recognizes and/or binds to a particular molecule (e.g., surface antigen, receptor) on the surface of circulating tumor cells, but does not recognize and bind to other unrelated molecules.

As used herein, the term "Circulating Tumor Cells (CTCs)" refers to cancer (tumor) cells present in blood (peripheral blood) at a concentration that can diagnose, stage, prognose, etc. cancer.

Targeted molecules

The method of the invention is based mainly on binding molecule-oligo (poly) nucleotide probes, which specifically target specific molecules on the surface of the pathogenic cells. The oligonucleotide probe is used as an amplification template for subsequent PCR quantitative analysis. The binding molecules label the pathogenic cells by binding to specific molecules on the surface of the pathogenic cells. The oligonucleotide is used for quantitatively detecting the ligand through a quantitative PCR technology so as to achieve the aim of quantifying the cells to be detected.

The targeting molecule of the invention is mainly based on the following structure: X-Y;

wherein X represents a specific high affinity molecule (usually Kd) for the pathogenic cell<10^-6mol/L), such as antibodies, high affinity polypeptide fragments, high affinity chemical small molecules, etc., for specifically binding to a particular molecule on the pathogenic cell; and Y is used as an amplification template for subsequent PCR quantitative analysis and is used for quantitative detection of target pathogenic cells.

X is a key molecule for targeting, which is used to identify and/or bind to a pathogenic cell, and therefore, it is generally set according to the type of pathogenic cell to be detected, for example, folic acid can identify and bind to a folate receptor (folate receptor) on the surface of a lung cancer cell; the monoclonal antibody against Her2 can recognize and bind to Her2 antigen on the surface of breast cancer cells; LHRH polypeptides can recognize and bind to the luteinizing hormone releasing hormone receptor (lhrhreceiver) on the surface of prostate cancer cells.

Currently, there has been intensive research on specific molecules present on the surface of various pathogenic cells, and various binding molecules against these surface molecules, including antibodies, ligands, polypeptides, small chemical molecules, and the like, have also been developed. The present invention is not particularly limited to binding molecules, and these binding molecules can be used in the method of the present invention to design various targeting molecules against pathogenic cells.

Y is an oligonucleotide molecule with a certain length, a specific primer can be designed based on the oligonucleotide molecule so as to realize PCR amplification, the detection limit is reduced through an amplification effect, and the detection sensitivity is improved. The oligonucleotide molecule may be double-stranded or single-stranded. The sequence of the oligonucleotide is not particularly limited, and preferably it is not complementary to the gene sequence of the human or animal body, and methods for designing and synthesizing the same are well known to those skilled in the art. The length of the oligonucleotide may preferably be 10-150bp (double-stranded) or 10-150nt (single-stranded).

The invention also includes modified oligonucleotide molecules obtained by means such as nucleic acid chain-based backbone modification techniques, said modifications not substantially altering the binding properties of the oligonucleotide molecules; those modifications which increase the stability of the oligonucleotide molecule are preferred. For example, the modification is a thio modification, or an alkyl modification at the 2' position of the ribose. It is understood that any modification capable of maintaining the binding properties of the oligonucleotide molecule is encompassed by the present invention.

There are various methods for modifying the backbone of an oligonucleotide, including a thio method in which the oxygen atoms of the phosphate bonds of the DNA backbone are replaced with sulfur atoms, and the thio may be all of the phosphate bonds or a part of the phosphate bonds. Modification of the thio group can greatly enhance the stability of the oligonucleotide molecule, thereby being beneficial to obtaining accurate detection results.

Preferably, X and Y are linked by a chemical covalent bond.

As a preferred mode of the present invention, the present inventors have developed targeting molecules for detecting Circulating Tumor Cells (CTCs) after repeated studies. This is because folate can recognize and bind to folate receptors (folate receptors) on the surface of tumor cells. In the targeting molecule, X is folic acid or a folate-cysteine conjugate. Compared with natural folic acid, the conjugate of folic acid and cysteine has improved affinity with folic acid receptor; furthermore, linking folic acid to cysteine significantly increases the solubility of folic acid, thereby increasing the amount of folic acid that contacts circulating tumor cells. The preparation of the folate-cysteine conjugate can be performed using techniques known in the art. The number of cysteines attached to folic acid may be one or more, e.g. 1-3, in which range cysteines may provide a good hydrophilic environment for folic acid. In the targeting molecule, the sequence of Y is diverse and is thio or non-thio.

Non-specific signal monitoring oligonucleotide probes

The inventor finds that in the process of detecting by using the targeting molecule, the oligonucleotide probe on the targeting molecule can be non-specifically combined with some DNA receptors on cells, and the like, so that the detection accuracy is reduced.

Therefore, after intensive research, the inventors designed an oligonucleotide fragment as a nonspecific signal monitoring oligonucleotide probe, which has a length and GC% similar to those of an oligonucleotide probe portion for detecting a targeting molecule of a pathogenic cell (difference of 30% or less), and can monitor nonspecific signals generated by the binding of the oligonucleotide portion of the targeting molecule to cells.

The non-specific signal monitoring oligonucleotide probe provided by the invention has similarity with the oligonucleotide probe on the targeting molecule, and the non-specific binding performance of the two on cells is similar. Thus, the non-specific binding of the oligonucleotide probe on the targeting molecule can be known by examining the occurrence of non-specific binding of the "non-specific signal-monitoring oligonucleotide probe" on the cell, so as to remove the interference of such non-specific binding with the detection result.

Reagent kit

The invention provides a kit for detecting pathogenic cells, which comprises: detecting a targeting molecule of the pathogenic cell; and non-specific signal monitoring oligonucleotide probes.

The primer matched with the targeting molecule comprises: the forward and reverse sequences are complementary with the sequences at the two ends of the targeting molecule, so that the targeting molecule can be used as a template to carry out PCR amplification, and the amplification of a detection result is realized.

If the oligonucleotide part of the targeting molecule is short (e.g. <50bp), in order to overcome the defect that the sequence is too short to perform PCR amplification, an extension primer can be designed for the probe on the targeting molecule, one end of the extension primer is complementary to the sequence of one end of the oligonucleotide probe on the targeting molecule, and the DNA is extended under the condition suitable for complementary DNA binding and extension to obtain the oligonucleotide comprising the original oligonucleotide probe sequence and the extension primer complementary sequence. And carrying out PCR amplification by using the lengthened oligonucleotide as a template to obtain a PCR qualitative or quantitative result of the template, thereby obtaining the PCR qualitative or quantitative result of the micro oligonucleotide.

The amplification is prepared by forward and reverse primers based on the sequences at both ends of the oligonucleotide obtained by the extension reaction of the extended primer and the micro-oligonucleotide.

And the extension primer covers the region with different bases in the oligonucleotide probe and the non-specific signal monitoring oligonucleotide probe sequence on the targeting molecule. That is, the extended sequence obtained by using the extended primer can represent the difference in sequence between the oligonucleotide probe and the non-specific signal monitoring oligonucleotide probe.

Preferably, the kit further comprises a fluorescent probe for fluorescent PCR, such as TaqMan probe, so as to facilitate quantitative analysis of PCR products.

In the kit, the targeting molecule, the nonspecific signal monitoring oligonucleotide probe and the matched primer or fluorescent probe thereof are independently filled in a suitable container, and preferably, the targeting molecule, the nonspecific signal monitoring oligonucleotide probe and the matched primer or fluorescent probe thereof are also prepared into a suitable dosage or concentration.

The kit may further comprise other reagents, such as reagents for PCR amplification (e.g., DNA polymerase, Realtime PCR Master Mix), buffers (e.g., PBS), washing solutions, and the like.

The kit may further comprise: instructions for use of the kit may be provided to instruct the skilled artisan.

Applications of

The invention also relates to a method for detecting pathogenic cells by using the kit, wherein the method can be used for diagnosing diseases, such as prognosis for patients or judging whether suspected people suffer from diseases; or for purposes other than diagnosis of disease, e.g., purely scientific research, or purely cellular typing (e.g., to distinguish between classes of tumor cells).

The method for detecting the pathogenic cells comprises the following steps: (1) blending the targeting molecule and the nonspecific signal monitoring oligonucleotide probe with a sample to be detected, and incubating; (2) collecting cells after incubation is finished, and respectively separating the targeting molecules and the nonspecific signal monitoring oligonucleotide probes from the cells; (3) performing PCR quantitative analysis by using the targeting molecules separated in the step (2) as templates to obtain a quantitative analysis result 1; performing PCR quantitative analysis by using the nonspecific signal monitoring oligonucleotide probe Y' separated in the step (2) as a template to obtain a quantitative analysis result 2; (4) and (4) subtracting the quantitative value of the quantitative analysis result 2 from the quantitative value of the quantitative analysis result 1 obtained in the step (3) to obtain the existence condition and the existence quantity of the pathogenic cells in the sample to be detected.

The PCR is preferably a fluorescent quantitative PCR technology, so that preferably, a fluorescent probe is also added into a PCR amplification system, so that analysis can be conveniently realized by a specific quantitative PCR instrument; more preferably, the fluorescent probe is a TaqMan probe.

As a preferred mode of the present invention, a control group and/or a standard group, i.e., a group in which the number of the folate-cysteine-oligonucleotide probes is known, are also provided, and a series of standards in different numbers are usually provided to constitute the standard group, whereby the presence and amount of pathogenic cells in a sample to be tested are analyzed.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Biological material and apparatus

H-Cys (Trt) -2-Cl Trt resin, HBTU and HOBT were all purchased from Novabiochem (U.S.A.).

PD-10 columns (SephadexG 25M) were purchased from general electric healthcare (USA).

SMCC (succinimide-4-cyclohexane-1-carbonate) was purchased from ThermoScientific (u.s.a.).

Erythrocyte lysates were purchased from solibao organisms (beijing, china).

Amino-modified single-stranded oligonucleotides, PBS, leukapheresis beads, Taqman MGB probes were purchased from Life technologies.

Primers used for PCR were purchased from Biotechnology engineering (Shanghai) Ltd.

The quantitative PCR detection reagent Realtime PCR Master Mix was purchased from TOYOBO, Japan.

The quantitative PCR instrument is a 7300realtime PCR system from Applied Biosystems.

The KB cell line was purchased from the shanghai cell bank of the chinese academy.

The remaining reagents were purchased from Sigma Aldrich.

Example 1 preparation of targeting molecules

In the following examples, the cells of interest were Circulating Tumor Cells (CTCs) and tumor patient samples containing CTCs were simulated by incorporating KB-pattern cells (human oral epidermoid carcinoma cells) into healthy human samples. In the targeting molecule with the structure of X-Y, the binding molecule X specifically targeting CTC is folic acid or a folic acid-cysteine conjugate.

The preparation method of the targeting molecule comprises the following steps:

first, a folate-cysteine conjugate is prepared. 270mg of H-Cys (Trt) -2-Cl Trt resin (1 equivalent based on the number of surface molecules of the resin) was immersed in 5mL of dimethylformamide for 30 minutes. 4 equivalents of Fmoc-Glu-OtBu (available from Novabiochem, USA) and 4 equivalents of N, N diisopropylethylamine were added, and 2.5 equivalents of HOBT and 2.5 equivalents of HBTU were added for 1 hour. Thereafter, 20% pyridine was added for 15 minutes to remove the protecting group Fmoc, and then the resin was washed 3 times with 5mL dimethylformamide. Thereafter, 4 equivalents of pteroic acid and 4 equivalents of N, N diisopropylethylamine and 2.5 equivalents of HOBT and HBTU were added and reacted overnight. The next day, the resin was washed 3 times with dimethylformamide, dichloromethane and methanol, respectively. The resin was dried with nitrogen for 60 minutes. Thereafter, a mixture of 10mL of trifluoroacetic acid, water and triisopropylsilane (volume ratio, 95:2.5:2.5) was added to the resin and reacted for 2 hours. The liquid reaction was collected in a round bottom flask and the liquid was evaporated on a rotary evaporator to approximately 1 mL. 50mL of precooled ether was added and the crude solid collected by centrifugation at 4000 g. The collected crude product was purified by HPLC to give 52mg of a 91% pure conjugate of folic acid (as gamma folic acid) -cysteine. HPLC analysis spectroscopy and mass spectrometry analysis were performed to confirm that the conjugate was obtained with a certain purity.

SMCC was dissolved in 500uL of a mixture of water and dimethyl sulfoxide (volume ratio 1:1) and then added to a single-or double-stranded oligonucleotide (PO16 or PO80, SEQ ID NO:1 or SEQ ID NO:2 in Table 1) at a concentration of 1mg/mL and incubated at room temperature for 4 hours. This reaction covalently bonds the amino group on the oligonucleotide to SMCC-N. The reacted product was purified by removing excess SMCC using a PD-10 column (SephadexG-25M). Then, the folic acid-cysteine conjugate was added to the reaction system to a final concentration of 1 mg/mL. The reaction is carried out by bonding the sulfenyl of folic acid-cysteine and maleimide on SMCC through a C-S covalent bond. The final product was purified by HPLC to obtain a folate-cysteine-oligonucleotide probe (F-PO16 or F-PO 80).

Example 2 targeting molecules can generate non-specific "background" signals

3mL of healthy human blood samples were taken and spiked with 20 and 0 KB cells, respectively. The experimental procedure was as follows:

(1) the blood sample is added with erythrocyte lysate and leukocyte removal magnetic beads, and erythrocytes and most leukocytes are removed. Dividing the sample into two groups, adding 10nM targeting molecule (F-PO16 or F-PO80 probe) into one group, mixing with the rest cell concentrate, and incubating for 40 min; the other group was incubated with 10nM oligonucleotide not linked to folate (PO16 or PO80 probe, SEQ ID NO:1 or 2) mixed with the remaining cell concentrate for 40 min;

(2) collecting cells after incubation, washing with PBS to remove unbound free probe molecules, and eluting the cell surface bound targeting molecules and the oligonucleotide not connected with folic acid with specific eluent respectively;

(3) and (3) respectively taking the target molecules eluted in the step (2) and the oligonucleotides not connected with the folic acid as templates, and performing fluorescent quantitative PCR amplification by using corresponding primers and fluorescent probes in the table 1(SEQ ID NO:3-9) to obtain detection signals.

TABLE 1

Note: PO represents the oligonucleotide portion of the targeting molecule, the following numbers represent the length of the oligonucleotide; RT represents an extension primer, and a target of PO16 is added into a PCR system, FP represents an amplification forward primer, RP represents an amplification reverse primer, and Taqman represents a Taqman MGB probe.

The results are shown in FIG. 1, and samples that did not incorporate KB cells, labeled with oligonucleotide ligated or not ligated with folate, were found to have no significant difference. In contrast, for the KB cell spiked samples, the detection values for the samples labeled with folate-oligonucleotides were significantly higher than for the samples labeled with non-folate-linked oligonucleotides. This result suggests that non-specific "background" signals can be generated due to binding of the oligonucleotide portion of the targeting molecule to the residual blood cells in the enriched sample, affecting the accuracy of the assay.

Example 3 selection of non-specific Signal monitoring oligonucleotide Probe Length

A healthy human blood sample is taken, and the experimental steps are as follows:

(1) adding erythrocyte lysate and leukocyte removing magnetic beads, and removing erythrocytes and most leukocytes. 10nM oligonucleotide probe (PO16 or PO80) and nonspecific signal monitoring oligonucleotide probe of different lengths (SEQ ID NO:10-13 for PO16, or SEQ ID NO:14-17 for PO80) were added separately, blended with the remaining cell concentrate, and incubated for 40 min;

(2) collecting cells after incubation, washing with PBS to remove unbound free probe molecules, and eluting the oligonucleotide probes bound to the cell surface and the non-specific signal monitoring oligonucleotide probes with specific eluents respectively;

(3) and (3) taking the oligonucleotide probe eluted in the step (2) or the non-specific signal monitoring oligonucleotide probe as a template, and respectively applying corresponding primers and fluorescent probes in the table 2 (aiming at PO16, SEQ ID NO:3-6) and the table 3 (aiming at PO80, SEQ ID NO:7-9) to perform fluorescent quantitative PCR amplification to obtain a detection signal.

The results are shown in Table 4.

TABLE 2

Note: QC represents the non-specific signal monitoring oligonucleotide probe, the following numbers represent the length of the oligonucleotide.

TABLE 3

TABLE 4

The results show that when the length difference between the QC probe and the PO probe is less than or equal to 30%, the difference between the detection values of the QC probe and the PO probe is less than or equal to 20%.

Example 4 selection of GC% of non-specific Signal monitoring oligonucleotide probes

(1) adding erythrocyte lysate and leukocyte removing magnetic beads, and removing erythrocytes and most leukocytes. 10nM oligonucleotide probe (PO16 or PO80) and non-specific signal monitoring oligonucleotide probe at different GC% were added separately (SEQ ID NO:18-22 for PO16, or SEQ ID NO:36-40 for PO80), blended with the remaining cell concentrate and incubated for 40 min;

(3) and (3) taking the oligonucleotide probe eluted in the step (2) or the non-specific signal monitoring oligonucleotide probe as a template, and respectively applying corresponding primers and fluorescent probes in the tables 1(SEQ ID NO:3-9), 5(SEQ ID NO:23-35) and 6(SEQ ID NO:41-48) to perform fluorescent quantitative PCR amplification to obtain a detection signal.

The results are shown in Table 7.

TABLE 5

TABLE 6

TABLE 7

The results show that when the GC% difference between the QC probe and the PO probe is less than or equal to 30%, the difference between the detection values of the QC probe and the PO probe is less than or equal to 20%.

Example 5 Effect of background values caused by non-specific binding of oligonucleotide moieties in targeting molecules on Signal to noise ratio

Collecting healthy human blood sample, and respectively treating under normal storage condition (control, storage temperature of 2-8 deg.C, and storage time within 24 hr) or abnormal storage condition (such as room temperature, or storage time over 24 hr). Another sample shows that the number of leukocytes is remarkably increased (more than or equal to 1.2X 10) due to the existence of inflammatory reaction⁷mL) of a blood sample from a benign patient. 3mL of each blood sample was taken and 20 and 0 KB cells were spiked. The experimental procedure was as follows:

(1) adding erythrocyte lysate and leukocyte removing magnetic beads, and removing erythrocytes and most leukocytes. Dividing the sample into two groups, adding 10nM targeting molecule (F-PO16 probe) and mixing with the rest cell concentrate, and incubating for 40 min; adding 10nM targeting molecule (F-PO16 probe) and 10nM nonspecific signal monitoring oligonucleotide probe (QC16-3 probe) into the other group, mixing with the rest cell concentrate, and incubating for 40 min;

(2) collecting cells after incubation, washing with PBS to remove unbound free probe molecules, and eluting the cell surface bound targeting molecules and nonspecific signal monitoring oligonucleotide probes with specific eluents respectively;

(3) taking the target molecules eluted in the step (2) as a template, and performing PCR amplification by using a primer (SEQ ID NO:3-6) of an oligonucleotide probe specifically aiming at the target molecules to obtain a total detection signal;

(4) and (3) taking the non-specific signal monitoring oligonucleotide probe molecules eluted in the step (2) as a template, and carrying out PCR amplification by using a primer and a fluorescent probe (SEQ ID NO:29, 4-6) which are specific to the molecules to obtain a background detection signal.

And subtracting the background value from the total detection value to obtain the detection value of the sample. The ratio of the sample spiked with 20 cells to the sample spiked with no cells was calculated as the signal-to-noise ratio.

The results are shown in Table 8.

TABLE 8

The above results show that the blood sample is preserved for more than 24 hours, the preservation temperature is not proper, or the number of white blood cells is higher than the normal value, which results in higher detection value. After background values caused by non-specific binding of oligonucleotide parts in the targeting molecules are deducted, the signal-to-noise ratio can be obviously improved.

Example 6 quantitative determination accuracy verification of F-PO16 Probe and QC Probe

F-PO16 probe and QC16-3 probe were diluted sequentially to six concentration gradients as calibrators, and the dilutions were used as negative controls. The primers, extension primers and fluorescent probes (SEQ ID NO:3-6, 29) aiming at the two probes are respectively used for carrying out fluorescent quantitative PCR amplification, and the performances of the two probe PCR quantitative systems, including linearity, amplification efficiency and amplification specificity (difference value of Ct value of negative control and PCR calibrator 1) and whether cross reaction exists between the two detection systems, are examined. The amplification reaction system is shown in Table 9.

TABLE 9

Composition of	Dosage of
		Realtime PCR Master Mix	12.5μL
Amplification primers	300nM
		Probe needle	100nM
Amplification template	2.5μL
		ddH₂O	Make up to 25 mu L

The reaction sequence is shown in Table 10, and fluorescence signals were detected during annealing at 35 ℃ in step 6, and FAM was selected for detection of fluorescence and corrected by ROX.

Watch 10

The results are shown in Table 11.

TABLE 11

The results show that the linear correlation coefficient R of the PCR quantitative standard curves of the F-PO16 probe and the QC16-3 probe is not less than 0.98, the amplification specificity and the amplification efficiency are high, cross reaction does not exist in the two detection systems, and the accuracy of quantitative detection is guaranteed.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A kit for detecting a pathogenic cell, comprising:

detecting targeting molecules of pathogenic cells, wherein the structure is X-Y; wherein X represents a binding molecule that specifically targets the pathogenic cell; y represents an oligonucleotide probe; "-" represents a covalent linkage, coupling or coupling relationship between X and Y;

the non-specific signal monitoring oligonucleotide probe Y ', Y' has a sequence length and sequence information similar to those of Y but different from those of Y in a partial base; wherein, the difference of the sequence length of Y ' and Y is less than or equal to 30 percent, the difference of GC percent in the sequence of Y ' and Y is less than or equal to 30 percent, and the sequence length of Y or Y ' is 10-150bp or 10-150 nt.

2. The kit of claim 1, wherein the sequence length of Y' is 11-120bp or 11-120 nt.

3. The kit of claim 1, wherein X is selected from the group consisting of: an antibody, ligand, chemical small molecule or polypeptide that specifically targets a pathogenic cell.

4. The kit of claim 3, wherein the pathogenic cell is a tumor cell and X is folate or a folate-cysteine conjugate.

5. The kit of claim 4, wherein said tumor cell is a circulating tumor cell.

6. The kit of claim 1, further comprising:

an extension primer aiming at Y, wherein the sequence structure of the extension primer is as follows: A-B; wherein, A is a random sequence, and the B sequence is complementary with the sequence at one end of the Y sequence; and

an extension primer aiming at Y', wherein the sequence structure of the extension primer is as follows: A-B'; wherein A is as defined above and the B 'sequence is complementary to the sequence at one of the ends of the Y' sequence.

7. The kit of claim 6, further comprising: a detection reagent that specifically detects an extended sequence comprising the Y and Y' sequences.

8. The kit of claim 7, wherein the detection reagent that specifically detects the extended sequence comprising the Y and Y' sequences is a fluorescent quantitative PCR detection reagent.

9. The kit of claim 8, wherein the fluorescent quantitative PCR detection reagent comprises: a forward primer, a reverse primer and a specific fluorescent probe that specifically amplify the extended sequence comprising the Y and Y' sequences.

10. Use of a kit according to any one of claims 1 to 9 for the non-diagnostic in vitro detection of pathogenic cells.

11. A method for non-diagnostically detecting a pathogenic cell in vitro, comprising:

(1) providing the kit of any one of claims 1-9, blending the targeting molecule and the non-specific signal monitoring oligonucleotide probe Y' with a sample to be tested, and incubating;

(2) collecting cells after incubation, and separating the targeting molecule of claim 1 and the non-specific signal monitoring oligonucleotide probe Y' from the cells respectively;